a, Phylogenetic profiling of BRAF and KSR1 domains demonstrates conservation of the BRS and CC-SAM domains throughout metazoan evolution. A concomitant loss of both domains is observed in the nematode lineage (indicated by ‘domain loss’). The conservation of each of BRAF and KSR1 domains with its human orthologue was colour-coded by BLAST E-value. RPS-BLAST was used to assess conservation of the CC-SAM domain. Conservation of the BRS was established by calculating the sum of the COILS score of amino acids located upstream of the RBD in each BRAF orthologue. b, Sequence alignment of the BRAF-specific (BRS) sequence from 14 metazoan species shows a conserved pattern of hydrophobic residues consistent with a coiled-coil region. Secondary structure elements identified in the BRS crystallographic structure are delineated above the sequence alignment. The heptad repeat (AbcDefg) typical in coiled-coil domains is shown below the alignment: positions A and D usually comprise hydrophobic amino acids while positions b, c, e, f, and g usually comprise polar or charged residues. BRS mutations used in this study are indicated by arrowheads. c, Sequence alignment of the CC-SAM sequence from 15 metazoan species. Secondary structure elements identified in the apo and BRS-bound CC-SAM structures are delineated above the sequence alignment. CC-SAM mutations used in this study are indicated by arrowheads below the alignment. Species in the phylogenetic tree and the sequence alignments are Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Strongylocentrotus purpuratus, Saccoglossus kowalevskii, Lingula anatina, Lottia gigantea, Drosophila melanogaster, Anopheles gambiae, Haemonchus contortus, Dictyocaulus viviparus, Necator americanus, Caenorhabditis briggsae, Caenorhabditis brenneri, Caenorhabditis remanei, Caenorhabditis elegans, Loa loa, Brugia malayi, Bursaphelenchus xylophilus, Trichinella papuae, Trichuris trichiura, Trichuris suis, Nematostella vectensis, Amphimedon queenslandica, and Trichoplax adherens. d, Y2H mating matrix of BRAF and KSR1 NTR domains and chimaeric fusions (the dashed box indicates the interaction between isolated BRS and CC-SAM domains). e, The BRS and CC-SAM domains specifically interact as determined by BRET titration experiments. In contrast, the BRS–BRS BRET pair did not generate a saturatable BRET signal. f, Swapping of the BRS and CC-SAM domains supports the BRAF–KSR1 interaction and transactivation of BRAF by KSR1. Fusing the BRS domain to CRAF NTR stimulates its interaction with KSR1NTR by co-IP (g) and by Y2H (h). Experiments in d–h were repeated at least three times. For gel source data, see Supplementary Fig. 1.